As part of a program in which the enzymes that participate in the process of detoxication are being examined as to their catalytic mechanism, attention is being concentrated on a sulfotransferase (aryl sulfotransferase IV). The enzyme catalyzes the transfer of the sulfuryl group from 3-phosphoadenosine 5-phosphosulfate (PAPS) to a very broad range of phenols; the products are 3-phosphoadenosine 5-phosphate (PAP) and the corresponding phenol sulfate. This laboratory has cloned the sulfotransferase from rat liver mRNA and can express it in very large quantities in Escherichia coli. In purifying the recombinant enzyme, however, two distinct forms of the sulfotransferase were physically separable. Labeled as forms A and B, both catalyzed the reverse reaction, i.e. the formation of free phenol from the phenol sulfate and PAP, but only form B could catalyze the physiological reaction. Further investigation revealed that form A had PAP bound to it so tightly that it would not allow the physiological donor, PAPS, to bind to the enzyme. The data suggested that protein cysteine plays a role in the formation of the two forms since PAP can be released by oxidation of protein thiols. The sulfotransferase consists of two subunits of identical amino acid constitution, each of which has five cysteine residues. Mutants have now been obtained for each cysteine in which that residue has been converted to serine. At present, the role of each of the cysteines in the folding of the protein and the binding of PAP is being dissected. Control of the oxidation state of the sulfotransferase and enzyme activity, can be regulated by the redox potential of the enzyme solution.